Methods of treating malignant lymphoproliferative disorders

ABSTRACT

Methods of treating malignant lymphoproliferative disorders in a patient, comprising administering an effective amount of a GSK-3β inhibitor, for example 9-ING-41, are provided. Also provided are methods for treating malignant lymphoproliferative disorders comprising administering a ({umlaut over (ι)}8K-3β inhibitor, for example 9-ING-41, in combination with a second or multiple therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States National Stage Application ofInternational Patent Application No. PCT/US2019/035576, filed Jun. 5,2019 which claims the benefit of priority to U.S. ProvisionalApplication No. 62/680,739, filed Jun. 5, 2018. The entirety of each ofthe aforementioned applications is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to methods of using GSK-3β inhibitors,including3-(5-Fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione,for treatment of malignant lymphoproliferative disorders.

BACKGROUND

Malignant lymphoproliferative disorders are a group of disorderscharacterized by the abnormal proliferation of lymphocytes. Two generaltypes of malignant lymphoproliferative disorders are malignant B-celllymphoproliferative disorders and malignant T-cell lymphoproliferativedisorders.

Malignant B-cell lymphoproliferative disorders include Diffuse largeB-cell lymphoma, acute lymphocytic leukemia, lymphoid blastic phaseChronic Myeloid Leukemia, Chronic lymphocytic leukemia/Small lymphocyticlymphoma, Extranodal marginal zone B-cell lymphomas, Mucosa-associatedlymphoid tissue lymphomas, Follicular lymphoma, Mantle cell lymphoma,Nodal marginal zone B-cell lymphoma, Burkitt lymphoma, Hairy cellleukemia, Primary central nervous system lymphoma, Splenic marginal zoneB-cell lymphoma, Waldenstrom's macroglobulinemia/Lymphoplasmacyticlymphoma, Multiple myeloma, Plasma cells dyscrasias, Plasma cellneoplasms, Primary mediastinal B-cell lymphoma, Hodgkin Disease, andCastelman's Disease.

Malignant T-cell lymphoproliferative disorders include T-cellleukemia/lymphoma, Extranodal natural killer/T-cell lymphoma, CutaneousT-cell lymphoma, Enteropathy-type T-cell lymphoma, AngioimmunoblasticT-cell lymphoma, Anaplastic large T/null-cell lymphoma, Subcutaneouspanniculitis-like T-cell lymphoma, T-cell acute lymphocytic leukemia,T-cell large granular lymphocyte leukemia, Lymphoid blastic phaseChronic Myeloid Leukemia, post-transplantation lymphoproliferativesyndromes, human T-cell leukemia virus type 1-positive (HTLV-1⁺) adultT-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL),and unspecified T-cell lymphoma.

Glycogen synthase kinase-3 (GSK-3) is a serine (S)/threonine (T) kinaseinitially described as a key regulator of metabolism, specificallyglycogen biosynthesis. Embi N, et al. Glycogen synthase kinase-3 fromrabbit skeletal muscle. Separation from cyclic-AMPdependent proteinkinase and phosphorylase kinase. Eur J Biochem. 1980; 107:519-27. It hassince been shown to play a role in several disease processes, includingcancer and aging, immune disorders, metabolic disorders, andneurological disorders through modulation of a large and diverse numberof substrates. Sutherland C. What Are the bona fide GSK3 Substrates? IntJ Alzheimers Dis. 2011; 2011:505607; Gao C, et al. GSK3: a key targetfor the development of novel treatments for type 2 diabetes mellitus andAlzheimer disease. Rev Neurosci. 2011; 23:1-11; Wang H, et al.,Convergence of the mammalian target of rapamycin complex 1- and glycogensynthase kinase 3-beta-signaling pathways regulates the innateinflammatory response. J Immunol. 2011; 186:5217-26; Klamer G, et al.Using small molecule GSK3beta inhibitors to treat inflammation. Curr MedChem. 2010; 17:2873-81; Henriksen E J. Dysregulation of glycogensynthase kinase-3 in skeletal muscle and the etiology of insulinresistance and type 2 diabetes. Curr Diabetes Rev. 2010; 6:285-93. GSK-3has two ubiquitously expressed and highly conserved isoforms, GSK-3α andGSK-3β, with both shared and distinct substrates and functional effects.Aberrant overexpression of GSK-3β has been shown to promote tumor growthand chemotherapy resistance in various solid tumors. Little is known,however, about the significance of GSK-3β in B-cell lymphomapathogenesis, resistance to therapy, and survival despite its knownfunction as a metabolic checkpoint regulator in B-cells. Jellusova J, etal. GSK3 is a metabolic checkpoint regulator in B cells. Nat Immunol.2017; 18:303-12.

GSK-3β inhibitors are of interest due to their ability to potentiallyalter the clinical course of diseases mediated by GSK-3β. Some GSK-3βinhibitors include tideglusib, LY2090314, 9-ING-41, CHIR-99021 andCHIR-98014, SB216763 and SB415286, AR-A011418, CG701338 and CG202796.See Amy Walz, Andrey Ugolkov, Sunandana Chandra, et al., MolecularPathways: Revisiting Glycogen Synthase Kinase-3b as a Target for theTreatment of Cancer, Clin Cancer Res; 23(8) Apr. 15, 2017, OF1-OF7.

3-(5-Fluorobenzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)pyrrole-2,5-dione(“9-ING-41”) is a GSK-3β inhibitor that has the following chemicalstructure:

The synthesis, properties, and/or biological activity of 9-ING-41 areset forth in U.S. Pat. No. 8,207,216; Gaisina et al., From a NaturalProduct Lead to the Identification of Potent and SelectiveBenzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3βInhibitors That Suppress Proliferation and Survival of Pancreatic CancerCells, J. Med. Chem. 2009, 52, 1853-1863; and Hilliard, et al., Glycogensynthase kinase 3β inhibitors induce apoptosis in ovarian cancer cellsand inhibit in-vivo tumor growth, Anti-Cancer Drugs 2011, 22:978-985.9-ING-41 has been reported to be useful for the treatment of certaincancers, including brain, lung, breast, ovarian, bladder, neuroblastoma,renal, and pancreatic cancers, as well as for treatment of traumaticbrain injury.

The clinical course for diffuse large B-cell lymphoma (DLBCL) remainsvariable despite improved response and survival with the addition of theanti-CD20 monoclonal antibody, rituximab, to standard chemotherapy inthe late 1990's. Coiffier B, et al. Long-term outcome of patients in theLNH-98.5 trial, the first randomized study comparing rituximab-CHOP tostandard CHOP chemotherapy in DLBCL patients: a study by the Grouped'Etudes des Lymphomes de l'Adulte. Blood. 2010; 116:2040-5; Feugier P,et al. Long-term results of the R-CHOP study in the treatment of elderlypatients with diffuse large B-cell lymphoma: a study by the Grouped'Etude des Lymphomes de l'Adulte. J Clin Oncol. 2005; 23:4117-26.Although 60% of patients enjoy long-term disease-free survival, a subsetof patients with adverse biology will have chemotherapy refractorydisease with less favorable outcomes. Sehn L H, et al. Introduction ofcombined CHOP plus rituximab therapy dramatically improved outcome ofdiffuse large B-cell lymphoma in British Columbia. J Clin Oncol. 2005;23:5027-33. In particular, dual translocation of c-MYC and BCL-2 inDLBCL, termed “double hit lymphoma” (“DHL”), is associated with pooroutcomes following standard R-CHOP (rituximab, cyclophosphamide,doxorubicin, vincristine, and prednisone), with few patients achievinglong-term survival. Petrich A M, et al. Impact of induction regimen andstem cell transplantation on outcomes in double-hit lymphoma: amulticenter retrospective analysis. Blood. 2014; 124:2354-61.

Thus, there exists a need for new methods for treating malignantlymphoproliferative disorders including lymphomas, and in particular fortreating therapy refractory lymphomas such as DLBCL.

SUMMARY

In some aspects, the present disclosure provides a method of treating amalignant lymphoproliferative disorder in a patient in need thereof,comprising administering to said patient an effective amount of a GSK-3βinhibitor.

In some aspects, the present disclosure provides a method of treating amalignant lymphoproliferative disorder in a patient in need thereof,comprising administering to said patient an effective amount of a GSK-3βinhibitor, wherein the malignant lymphoproliferative disorder is amalignant B-cell lymphoproliferative disorder.

In some aspects, the present disclosure provides a method of treating amalignant B-cell lymphoproliferative disorder in a patient in needthereof, comprising administering to said patient an effective amount ofa GSK-3β inhibitor, wherein the malignant B-cell lymphoproliferativedisorder is Diffuse large B-cell lymphoma, acute lymphocytic leukemia,lymphoid blastic phase Chronic Myeloid Leukemia, Chronic lymphocyticleukemia/Small lymphocytic lymphoma, Extranodal marginal zone B-celllymphomas, Mucosa-associated lymphoid tissue lymphomas, Follicularlymphoma, Mantle cell lymphoma, Nodal marginal zone B-cell lymphoma,Burkitt lymphoma, Hairy cell leukemia, Primary central nervous systemlymphoma, Splenic marginal zone B-cell lymphoma, Waldenstrom'smacroglobulinemia/Lymphoplasmacytic lymphoma, Multiple myeloma, Plasmacells dyscrasias, Plasma cell neoplasms, Primary mediastinal B-celllymphoma, Hodgkin Disease, or Castelman's Disease.

In some aspects, the present disclosure provides a method of treating amalignant B-cell lymphoproliferative disorder in a patient in needthereof, comprising administering to said patient an effective amount ofa GSK-3β inhibitor, wherein the malignant B-cell lymphoproliferativedisorder is Diffuse large B-cell lymphoma.

In some aspects, the present disclosure provides a method of treating aDiffuse large B-cell lymphoma in a patient in need thereof, comprisingadministering to said patient an effective amount of a GSK-3β inhibitor,wherein the Diffuse large B-cell lymphoma is Double-Hit lymphoma.

In some aspects, the present disclosure provides a method of treating amalignant lymphoproliferative disorder in a patient in need thereof,comprising administering to said patient an effective amount of a GSK-3βinhibitor, wherein the malignant lymphoproliferative disorder is amalignant T-cell lymphoproliferative disorder.

In some aspects, the present disclosure provides a method of treating amalignant T-cell lymphoproliferative disorder in a patient in needthereof, comprising administering to said patient an effective amount ofa GSK-3β inhibitor, wherein the malignant T-cell lymphoproliferativedisorder is T-cell leukemia/lymphoma, Extranodal natural killer/T-celllymphoma, Cutaneous T-cell lymphoma, Enteropathy-type T-cell lymphoma,Angioimmunoblastic T-cell lymphoma, Anaplastic large T/null-celllymphoma, Subcutaneous panniculitis-like T-cell lymphoma, T-cell acutelymphocytic leukemia, T-cell large granular lymphocyte leukemia,Lymphoid blastic phase Chronic Myeloid Leukemia, post-transplantationlymphoproliferative syndromes, human T-cell leukemia virus type1-positive (HTLV-1⁺) adult T-cell leukemia/lymphoma (ATL), T-cellprolymphocytic leukemia (T-PLL), or unspecified T-cell lymphoma.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the malignant lymphoproliferativedisorder is chemotherapy-refractory.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the a GSK-3β inhibitor is9-ING-41, tideglusib, LY2090314, CHIR-99021, CHIR-98014, SB216763,SB415286, AR-A011418, CG701338, or CG202796.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor is 9-ING-41,tideglusib, or LY2090314.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor is 9-ING-41.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor istideglusib.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor is LY2090314.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor isadministered in combination with a second therapeutic agent.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor isadministered in combination with a second therapeutic agent, and whereinthe second therapeutic agent is administered in a sub-therapeuticamount.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor isadministered in combination with a second therapeutic agent, wherein thesecond therapeutic agent is an anticancer agent.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with an anticancer agent, wherein the anticancer agent is anapoptosis modulator, a CDK modulator, or a modulator of themTOR/AKT/PI3K pathway.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with an anticancer agent, wherein the anticancer agent is anapoptosis modulator.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with an apoptosis modulator, wherein the an apoptosismodulator is a Bcl-2 inhibitor.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a Bcl-2 inhibitor, wherein the Bcl-2 inhibitor isvenetoclax, ABT-737, or navitoclax.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a Bcl-2 inhibitor, wherein the Bcl-2 inhibitor isvenetoclax.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor isadministered in combination with an anticancer agent, wherein theanticancer agent is a CDK modulator.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a CDK modulator, wherein the a CDK modulator is a CDK9inhibitor.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a CDK9 inhibitor, wherein the CDK9 inhibitor isBAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a CDK9 inhibitor, wherein the CDK9 inhibitor isBAY-1143572.

In some aspects, the present disclosure provides a method according toany one of the aspects above, wherein the GSK-3β inhibitor isadministered in combination with an anticancer agent, wherein theanticancer agent is a modulator of the mTOR/AKT/PI3K pathway.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a modulator of the mTOR/AKT/PI3K pathway, wherein themodulator of the mTOR/AKT/PI3K pathway is a PI3K inhibitor.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a PI3K inhibitor, wherein the a PI3K inhibitor iscopanlisib or idelalisib.

In some aspects, the present disclosure provides a method according tothe previous aspect, wherein the GSK-3β inhibitor is administered incombination with a PI3K inhibitor, wherein the PI3K inhibitor isidelalisib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IE show the viability and proliferation of lymphoma cells with9-ING-41 treatment. 10,000 cells (SUDHL-4 (FIG. 1B), KPUM-UH1 (FIG. 1C),Karpas 422 (FIG. 1D), TMD8 (FIG. 1E)) per 96-well plate well were leftuntreated or treated with 1 μM 9-ING-41 in triplicate, and the number ofcells on days 1, 3, 5 and 7 were calculated using the MTS assay.Briefly, 20 μL of MTS reagent was added to cells and incubated for 2hours and, the absorbance at 490 nm (A490) was read using a Biotek platereader. Absorbance at OD=490 nm increases proportionally to celldensity. Error bars represent std. deviation between replicates. Day 3viability is shown in FIG. 1A.

FIGS. 2A-2O show the viability of lymphoma cells with chemotherapyagents in combination with 9-ING-41. 10,000 cells (FIGS. 1A, F, K:Daudi; FIGS. 1B, G, L: SUDHL-4; FIGS. 1C, H, M: KPUM-UH1; FIGS. 1D, I,N: Karpas 422; E, J, O: TMD8) were plated per well of a 96-well plateand treated with a dose response series of both 9-ING-41 (0-0.5 μM) andVenetoclax (0-5,000 nM) (FIGS. 1A-E) or BAY-1143572 (0-50 μM) (FIGS.1F-J) or Idelalisib (0-50 μM) (FIGS. 1K-O) in triplicate. Viabilityafter 3 days was analyzed using the MTS assay. Briefly, 20 μl of MTSreagent was added to cells and incubated for 2 hours and, the absorbanceat 490 nm was read using a Biotek plate reader. Relative absorbance iscalculated after setting the average absorbance of the no-treatmentcontrol as 1. Absorbance at OD=490 nm increases proportionally to celldensity.

FIG. 3 shows IC₅₀ values for venetoclax, BAY1143572, and idelalisib withor without 0.5 μM 9-ING-41.

FIG. 4 shows that GSK3α and GSK3β mRNA and proteins are overexpressed inlymphoma: (A) Real-time PCR quantitation showing GSK3α and GSK3β mRNAsare overexpressed in lymphoma lines in comparison to low expression innormal B or T lymphocytes. (B) Western blot images demonstrating GSK3αand GSK3β proteins are also abundantly expressed in various lymphomalines in comparison to purified normal B or T lymphocytes.

FIG. 5 shows that GSK3 is essential for lymphoma cell proliferation andsurvival. Unstimulated peripheral blood B- and T-lymphocytes isolatedfrom a healthy donor were used as normal control. Pro-apoptotic effectof the GSK3 inhibitor 9-ING-41 in various MCL and TCL lines (A) andDLBCL lines (B). (C) Cell proliferation profile of various lymphoma celllines upon treatment with 9-ING-41. Results (A-C) are from 3 independentexperiments.

FIG. 6 shows that GSK3 inhibition or deletion in lymphoma cells leads tocell cycle arrest in G2/M. (A) Cell cycle profile of 3 representativecell lines Jeko, Mino, and OCI-Ly3 after 24 hour treatment with 0, 1.0and 2.0 μM 9-ING-41. (B) Cell cycle profiles of parental Ly-1 cells andGSK3α, GSK3β, GSK3αβ knockout subclones. Inset: Western blot imageshowing the depletion of GSK3α and GSK3β protein in the knockout Ly-1subclones.

FIG. 7 shows that inhibition of GSK3 by 9-ING-41 leads to mitoticprophase arrest. (A) Cartoon depiction of the sequential steps (M1-M5)during mitosis (purchased from Shutterstock and modified). (B).Representative Wright stain images of Jeko cells untreated and treatedwith 1.0 uM 9-ING-41 for 24 hours. Various mitotic stage cells (M1-M5)are readily identified in untreated cells (left panel) while onlyprophase (M1) cells in large number are seen in 9-ING-41 cells (rightpanel). (C). Bar chart showing the number of mitotic M1-M5 cellsidentified when 100 untreated or 9-ING-41 treated Jeko cells werecounted. Similar results (data not shown) were observed in at least 4different lymphoma cell lines.

FIG. 8 shows that GSK3β localized to centrosomes. (A) Animmunofluorescence image showing GSK3β is localized to the nucleus andcentrosome pairs in interphase Jeko cells. (B) A close-up(magnification) image of that shown in (A). (C-F) single ormulti-channel images of coimmunostaining of GSK3β and pericentrin inwild-type Ly-1 cells showing their colocalization to the centrosomes.(G-H) single or multi-channel images of co-immunostaining of GSK3β andpericentrin in GSK3β null in Ly-1 cells showing the staining is specificto GSK3β. (K) An immunofluorescence image showing GSK3β (green)localized to firework like structures resembling to mitotic spindles inmitotic Jeko cells. (L) A close-up image of a mitotic cell shown in (K).(M) An overlay image showing the microtubule structure by α-tubulin(red) staining and DNA (in blue). (N) A close-up image of that shown in(M). (O-P) Images showing that spindle structure staining of GSK3β isabsent in GSK3β null Ly-1 cells. (Q) An immunofluorescence image showingGSK3β localized to mitotic spindle structure and polarized centrosomesin 9-ING-41 treated Jeko cells. (R) A close-up image of a representativemitotic cell shown in (Q).

FIG. 9 shows aberrant expression of GSK3 proteins in primary lymphomapatient (P) cells and their proliferative response to 9-ING-41. (A)Immunoblot of GSK3α and GSK3β proteins showing overexpression in patientsamples vs normal B-cell control. P1: MCL; P2: High grade B-celllymphoma; P3: follicular large B-cell lymphoma 3B; P4: DLBCL; P5:angioimmunoblastic T-cell lymphoma. (B) 9-ING-41 inhibited proliferationin all 5 patient samples. (C) Immunohistochemistry staining of GSK3β onparaffin tissue sections of patients with various lymphoma.Representative images show the spectrum of GSK3β (in brown)overexpression in different lymphoma samples. Methylene bluecounterstaining (in blue) shows cells negative for GSK3β in thebackground and in antibody negative control panel. Images were collectedunder 40× magnification.

FIG. 10 shows anti-lymphoma effect of 9-ING-41 in vivo in Jeko derivedxenograft mouse model. (A) Experimental design showing 9-ING-41treatment schedule and dosage. (B) Bioluminescence images of xenograftbearing mice untreated or treated with 9-ING-41. The images shown werecollected at the end of the experiment (day 17). The experiment was donetwice both showed similar results.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present subject matter may be understood more readily by referenceto the following detailed description which forms a part of thisdisclosure. It is to be understood that this invention is not limited tothe specific methods, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

As employed above and throughout the disclosure, the following terms andabbreviations, unless otherwise indicated, shall be understood to havethe following meanings.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “acompound” is a reference to one or more of such compounds andequivalents thereof known to those skilled in the art, and so forth. Theterm “plurality”, as used herein, means more than one. When a range ofvalues is expressed, another embodiment incudes from the one particularand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it isunderstood that the particular value forms another embodiment. Allranges are inclusive and combinable.

As used herein, the terms “component,” “composition,” “composition ofcompounds,” “compound,” “drug,” “pharmacologically active agent,”“active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament”are used interchangeably herein to refer to a compound or compounds orcomposition of matter which, when administered to a subject (human oranimal) induces a desired pharmacological and/or physiologic effect bylocal and/or systemic action.

As used herein, the terms “treating”, “treatment” or “therapy” (as wellas different forms thereof) include preventative (e.g., prophylactic),curative or palliative treatment. As used herein, the term “treating”includes alleviating or reducing at least one adverse or negative effector symptom of a condition, disease or disorder. This condition, diseaseor disorder can be cancer.

As employed above and throughout the disclosure the term “effectiveamount” refers to an amount effective, at dosages, and for periods oftime necessary, to achieve the desired result with respect to thetreatment of the relevant disorder, condition, or side effect. It willbe appreciated that the effective amount of components of the presentinvention will vary from patient to patient not only with the particularcompound, component or composition selected, the route ofadministration, and the ability of the components to elicit a desiredresult in the individual, but also with factors such as the diseasestate or severity of the condition to be alleviated, hormone levels,age, sex, weight of the individual, the state of being of the patient,and the severity of the pathological condition being treated, concurrentmedication or special diets then being followed by the particularpatient, and other factors which those skilled in the art willrecognize, with the appropriate dosage being at the discretion of theattending physician. Dosage regimes may be adjusted to provide theimproved therapeutic response. An effective amount is also one in whichany toxic or detrimental effects of the components are outweighed by thetherapeutically beneficial effects.

In some embodiments, an effective amount is based on the weight of thepatient. In some embodiments, an effective amount of 9-ING-41 is aboutfrom 0.1 mg/kg to 10 mg/kg, for example, about 0.1 mg/kg, about 0.5mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, orabout 10 mg/kg.

As employed above and throughout the disclosure the term“sub-therapeutic amount” refers to an amount that is ineffective whenadministered as the sole therapeutic agent.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

Within the present invention, the disclosed compounds may be prepared inthe form of pharmaceutically acceptable salts. “Pharmaceuticallyacceptable salts” refer to derivatives of the disclosed compoundswherein the parent compound is modified by making acid or base saltsthereof. Examples of pharmaceutically acceptable salts include, but arenot limited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, such conventional non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Thesephysiologically acceptable salts are prepared by methods known in theart, e.g., by dissolving the free amine bases with an excess of the acidin aqueous alcohol, or neutralizing a free carboxylic acid with analkali metal base such as a hydroxide, or with an amine.

Compounds described herein can be prepared in alternate forms. Forexample, many amino-containing compounds can be used or prepared as anacid addition salt. Often such salts improve isolation and handlingproperties of the compound. For example, depending on the reagents,reaction conditions and the like, compounds as described herein can beused or prepared, for example, as their hydrochloride or tosylate salts.Isomorphic crystalline forms, all chiral and racemic forms, N-oxide,hydrates, solvates, and acid salt hydrates, are also contemplated to bewithin the scope of the present invention.

Certain acidic or basic compounds of the present invention may exist aszwitterions. All forms of the compounds, including free acid, free baseand zwitterions, are contemplated to be within the scope of the presentinvention. It is well known in the art that compounds containing bothamino and carboxy groups often exist in equilibrium with theirzwitterionic forms. Thus, any of the compounds described herein thatcontain, for example, both amino and carboxy groups, also includereference to their corresponding zwitterions.

The term “administering” means either directly administering a compoundor composition of the present invention, or administering a prodrug,derivative or analog which will form an equivalent amount of the activecompound or substance within the body.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein, and refer to an animal, for example a human, towhom treatment, including prophylactic treatment, with thepharmaceutical composition according to the present invention, isprovided. The term “subject” as used herein refers to human andnon-human animals. The terms “non-human animals” and “non-human mammals”are used interchangeably herein and include all vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent, (e.g. mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, horses and non-mammals such as reptiles, amphibians,chickens, and turkeys.

The present disclosure provides a method of treating a malignantlymphoproliferative disorder in a patient in need thereof, comprisingadministering to said patient an effective amount of a GSK-3β inhibitor.

The malignant lymphoproliferative disorder that may be treated using themethods of the present disclosure may be a malignant B-celllymphoproliferative disorder, or a malignant T-cell lymphoproliferativedisorder. In some embodiments, the malignant lymphoproliferativedisorder is a malignant B-cell lymphoproliferative disorder. In otherembodiments, the malignant lymphoproliferative disorder is a malignantT-cell lymphoproliferative disorder.

In some embodiments, the malignant B-cell lymphoproliferative disorderthat may be treated using the methods of the present disclosure isDiffuse large B-cell lymphoma, acute lymphocytic leukemia, lymphoidblastic phase Chronic Myeloid Leukemia, Chronic lymphocyticleukemia/Small lymphocytic lymphoma, Extranodal marginal zone B-celllymphomas, Mucosa-associated lymphoid tissue lymphomas, Follicularlymphoma, Mantle cell lymphoma, Nodal marginal zone B-cell lymphoma,Burkitt lymphoma, Hairy cell leukemia, Primary central nervous systemlymphoma, Splenic marginal zone B-cell lymphoma, Waldenstrom'smacroglobulinemia/Lymphoplasmacytic lymphoma, Multiple myeloma, Plasmacells dyscrasias, Plasma cell neoplasms, Primary mediastinal B-celllymphoma, Hodgkin Disease, or Castelman's Disease.

In some embodiments, the malignant B-cell lymphoproliferative disorderthat is treated using the methods of the present disclosure is Diffuselarge B-cell lymphoma. In some embodiments, the Diffuse large B-celllymphoma is a Double-Hit lymphoma.

In some embodiments, the malignant B-cell lymphoproliferative disorderthat is treated using the methods of the present disclosure is MantleCell Lymphoma.

In some embodiments, the malignant T-cell lymphoproliferative disorderthat may be treated using the methods of the present disclosure isT-cell leukemia/lymphoma, Extranodal natural killer/T-cell lymphoma,Cutaneous T-cell lymphoma, Enteropathy-type T-cell lymphoma,Angioimmunoblastic T-cell lymphoma, Anaplastic large T/null-celllymphoma, Subcutaneous panniculitis-like T-cell lymphoma, T-cell acutelymphocytic leukemia, T-cell large granular lymphocyte leukemia,Lymphoid blastic phase Chronic Myeloid Leukemia, post-transplantationlymphoproliferative syndromes, human T-cell leukemia virus type1-positive (HTLV-1+) adult T-cell leukemia/lymphoma (ATL), T-cellprolymphocytic leukemia (T-PLL), or unspecified T-cell lymphoma.

In some embodiments, the malignant T-cell lymphoproliferative disorderthat is treated using the methods of the present disclosure is T-CellLymphoma.

In some aspects of the present disclosure, the malignantlymphoproliferative disorder may be a chemotherapy refractory malignantlymphoproliferative disorder. A chemotherapy refractory malignantlymphoproliferative disorder is a malignant lymphoproliferative disorderthat has failed to respond to one or more chemotherapeutic treatments.In some embodiments, the chemotherapy refractory malignantlymphoproliferative disorder is a double-hit lymphoma (i.e., a lymphomahaving dual translocation of c-MYC and BCL-2). In other embodiments, thechemotherapy to which the refractory malignant lymphoproliferativedisorder has failed to respond is R-CHOP (rituximab, cyclophosphamide,doxorubicin, vincristine, and prednisone). In yet other embodiments, thechemotherapy to which the refractory malignant lymphoproliferativedisorder has failed to respond is a combination of cytotoxic therapieswith or without monoclonal antibodies, kinase inhibitors, enzymemodulators, and apoptosis modifiers.

The patients whose conditions are treated using the methods of thepresent invention are animals, preferably mammals. In some embodiments,the patients are human beings. In other embodiments, the patients arecanine (i.e., dogs). In yet other embodiments, the patients are feline(i.e., cats). In preferred embodiments, the patients whose conditionsare treated using the methods of the present disclosure are humanbeings.

The GSK-3β inhibitor that is administered in the methods of the presentdisclosure is any compound that inhibits the activity of glycogensynthase kinase-3β (GSK-3β).

In some aspects, the GSK-3β inhibitor that is administered in themethods of the present disclosure is 9-ING-41, tideglusib, LY2090314,CHIR-99021, CHIR-98014, SB216763, SB415286, AR-A011418, CG701338, orCG202796.

In some embodiments, the GSK-3β inhibitor that is administered in themethods of the present disclosure is 9-ING-41. Thus, in some aspects,the present disclosure provides a method of treating a malignantlymphoproliferative disorder in a patient in need thereof, comprisingadministering to said patient an effective amount of 9-ING-41.

In other embodiments, the GSK-3β inhibitor that is administered in themethods of the present disclosure is tideglusib, and the presentdisclosure provides a method of treating a malignant lymphoproliferativedisorder in a patient in need thereof, comprising administering to saidpatient an effective amount of tideglusib.

In yet other embodiments, the GSK-3β inhibitor that is administered inthe methods of the present disclosure is LY2090314, and the presentdisclosure provides a method of treating a malignant lymphoproliferativedisorder in a patient in need thereof, comprising administering to saidpatient an effective amount of LY2090314.

In the methods of treating malignant lymphoproliferative disorders ofthe present disclosure, an effective amount of a GSK-3β inhibitor may beadministered to the patient as the sole therapeutic agent, or incombination with one or more other therapeutic agents.

In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering a GSK-3β inhibitoras the sole therapeutic agent. When the GSK-3β inhibitor is the soletherapeutically effective compound administered in the methods of thepresent disclosure, the treatment is referred to as a monotherapy. Insome embodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 as the sole therapeuticagent. In other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib as thesole therapeutic agent. In yet other embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administeringLY2090314 as the sole therapeutic agent.

In other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering a GSK-3β incombination with one or more other therapeutic agents. When the GSK-3βinhibitor is administered in combination with a second therapeuticagent, the treatment is referred to as combination therapy. Incombination therapy, it is not necessary that the GSK-3β inhibitor andthe second therapeutic agent be introduced into, or applied onto, thepatient's body simultaneously. Combination therapy requires only thatthe GSK-3β inhibitor and the second therapeutic agent be present in oron the patient's body at the same time. Thus, combination therapy doesnot imply any particular dosing schedule.

In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with one or more other therapeutic agents. In otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering tideglusib in combination with one ormore other therapeutic agents. In yet other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringLY2090314 in combination with one or more other therapeutic agents.

In some aspects, the method of treating malignant lymphoproliferativedisorders comprises administering a GSK-3β inhibitor in combination withan apoptosis modulator. In some embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administering 9-ING-41in combination with an apoptosis modulator. In other embodiments, themethod of treating malignant lymphoproliferative disorders comprisesadministering tideglusib in combination with an apoptosis modulator. Inyet other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering LY2090314 incombination with an apoptosis modulator.

In some aspects, the apoptosis modulator is a Bcl-2 inhibitor. ExemplaryBcl-2 inhibitors include venetoclax, ABT-737, and navitoclax. In someaspects, the method of treating malignant lymphoproliferative disorderscomprises administering a GSK-3β inhibitor in combination with a Bcl-2inhibitor. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with a Bcl-2 inhibitor. In other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringtideglusib in combination with a Bcl-2 inhibitor. In yet otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering LY2090314 in combination with a Bcl-2inhibitor.

In some aspects, the Bcl-2 inhibitor is venetoclax, ABT-737, ornavitoclax. In some aspects, the method of treating malignantlymphoproliferative disorders comprises administering a GSK-3β inhibitorin combination with venetoclax, ABT-737, or navitoclax. In someembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 in combination withvenetoclax, ABT-737, or navitoclax. In other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringtideglusib in combination with venetoclax, ABT-737, or navitoclax. Inyet other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering LY2090314 incombination with venetoclax, ABT-737, or navitoclax.

In some aspects, the Bcl-2 inhibitor is venetoclax. In other aspects,the Bcl-2 inhibitor is ABT-737. In yet other aspects, the Bcl-2inhibitor is navitoclax. In some aspects, the method of treatingmalignant lymphoproliferative disorders comprises administering a GSK-3βinhibitor in combination with venetoclax. In some aspects, the method oftreating malignant lymphoproliferative disorders comprises administeringa GSK-3β inhibitor in combination with ABT-737. In some aspects, themethod of treating malignant lymphoproliferative disorders comprisesadministering a GSK-3β inhibitor in combination with navitoclax.

In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with venetoclax. In some embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administering 9-ING-41in combination with ABT-737. In some embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administering 9-ING-41in combination with navitoclax.

In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with venetoclax. In some embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administeringtideglusib in combination with ABT-737. In some embodiments, the methodof treating malignant lymphoproliferative disorders comprisesadministering tideglusib in combination with navitoclax.

In yet other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering LY2090314 incombination with venetoclax. In yet other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringLY2090314 in combination with ABT-737. In yet other embodiments, themethod of treating malignant lymphoproliferative disorders comprisesadministering LY2090314 in combination with navitoclax.

In some aspects, the method of treating malignant lymphoproliferativedisorders comprises administering a GSK-3β inhibitor in combination witha cyclin-dependent kinase (CDK) modulator. In some embodiments, themethod of treating malignant lymphoproliferative disorders comprisesadministering 9-ING-41 in combination with a cyclin-dependent kinase(CDK) modulator. In other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with a cyclin-dependent kinase (CDK) modulator. In yet otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering LY2090314 in combination with acyclin-dependent kinase (CDK) modulator.

In some aspects, the CDK modulator is a cyclic dependent kinase 9(“CDK9”) inhibitor. Exemplary CDK9 inhibitor include BAY-1143572,LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032), AT7519,P276-00, AZD5438, PHA-767491, or PHA-793887. In some aspects, the methodof treating malignant lymphoproliferative disorders comprisesadministering a GSK-3β inhibitor in combination with a CDK9 inhibitor.In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with a CDK9 inhibitor. In other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringtideglusib in combination with a CDK9 inhibitor. In yet otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering LY2090314 in combination with a CDK9inhibitor.

In some aspects, the CDK9 inhibitor is BAY-1143572, LDC000067,Dinaciclib (SCH727965), SNS-032 (BMS-387032), AT7519, P276-00, AZD5438,PHA-767491, or PHA-793887. In some aspects, the method of treatingmalignant lymphoproliferative disorders comprises administering a GSK-3βinhibitor in combination with BAY-1143572, LDC000067, Dinaciclib(SCH727965), SNS-032 (BMS-387032), AT7519, P276-00, AZD5438, PHA-767491,or PHA-793887. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with BAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032(BMS-387032), AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887. Inother embodiments, the method of treating malignant lymphoproliferativedisorders comprises administering tideglusib in combination withBAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887. In yet otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering LY2090314 in combination withBAY-1143572, LDC000067, Dinaciclib (SCH727%5), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887.

In some aspects, the CDK9 inhibitor is BAY-1143572. In some aspects, themethod of treating malignant lymphoproliferative disorders comprisesadministering a GSK-3β inhibitor in combination with BAY-1143572. Insome embodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 in combination withBAY-1143572. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with BAY-1143572. In yet other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringLY2090314 in combination with BAY-1143572.

In some aspects, the method of treating malignant lymphoproliferativedisorders comprises administering a GSK-3β inhibitor in combination witha modulator of the MTOR/AKT/PI3 pathway. In some embodiments, the methodof treating malignant lymphoproliferative disorders comprisesadministering 9-ING-41 in combination with a modulator of theMTOR/AKT/PI3 pathway. In other embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administeringtideglusib in combination with a modulator of the MTOR/AKT/PI3 pathway.In yet other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering LY2090314 incombination with a modulator of the MTOR/AKT/PI3 pathway.

In some aspects, the a modulator of the MTOR/AKT/PI3 pathway is a PI3Kinhibitor. Exemplary PI3K inhibitors include copanlisib and idelalisib.In some aspects, the method of treating malignant lymphoproliferativedisorders comprises administering a GSK-3β inhibitor in combination witha PI3K inhibitor. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering 9-ING-41 incombination with a PI3K inhibitor. In other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringtideglusib in combination with a PI3K inhibitor. In yet otherembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering LY2090314 in combination with a PI3Kinhibitor.

In some aspects, the PI3K inhibitor is copanlisib or idelalisib. In someaspects, the method of treating malignant lymphoproliferative disorderscomprises administering a GSK-30 inhibitor in combination withcopanlisib or idelalisib. In some embodiments, the method of treatingmalignant lymphoproliferative disorders comprises administering 9-ING-41in combination with copanlisib or idelalisib. In other embodiments, themethod of treating malignant lymphoproliferative disorders comprisesadministering tideglusib in combination with copanlisib or idelalisib.In yet other embodiments, the method of treating malignantlymphoproliferative disorders comprises administering LY2090314 incombination with copanlisib or idelalisib.

In some aspects, the PI3K inhibitor is copanlisib. In some aspects, themethod of treating malignant lymphoproliferative disorders comprisesadministering a GSK-3β inhibitor in combination with copanlisib. In someembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 in combination withcopanlisib. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with copanlisib. In yet other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringLY2090314 in combination with copanlisib.

In some aspects, the PI3K inhibitor is idelalisib. In some aspects, themethod of treating malignant lymphoproliferative disorders comprisesadministering a GSK-3β inhibitor in combination with idelalisib. In someembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 in combination withidelalisib. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with idelalisib. In yet other embodiments, the method oftreating malignant lymphoproliferative disorders comprises administeringLY2090314 in combination with idelalisib.

In still other aspects, the second therapeutic agent is one or more of5-fluorouracil, abiraterone acetate, acetylcholine, ado-trastuzumabemtansine, afatinib, aldesleukin, alectinib, alemtuzumab, alitretinoin,aminolevulinic acid, anastrozole, anastrozole, aprepitant, arsenictrioxide, asparaginase Erwinia chrysanthemi, atezolizumab, axitinib,azacitidine, belinostat, bendamustine, benzyl isothiocyanate,bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab,bortezomib, bosutinib, brentuximab vedotin, busulfan, cabazitaxel,cabozantinib, capecitabine, carboplatin, carfilzomib, carmustine,ceritinib, cetuximab, chlorambucil, cisplatin, clofarabine, cobimetinib,crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine,dacarbazine, dactinomycin, daratumumab, dasatinib, daunorubicin,decitabine, defibrotide sodium, degarelix, denileukin diftitox,denosumab, dexamethasone, dexrazoxane, dihydrotestosterone (DHT),dinutuximab, docetaxel, doxorubicin, elotuzumab, eltrombopag,enzalutamide, epirubicin, eribulin mesylate, erlotinib, etoposide,everolimus, exemestane, exemestane, filgrastim, fludarabine phosphate,flutamide, fulvestrant, fulvestrant, gefitinib, gemcitabine, gemtuzumab,gemtuzumab ozogamicin, glucarpidase, goserelin acetate, hydroxyurea,ibritumomab tiuxetan, ibrutinib, idarubicin, idelalisib, ifosfamide,imatinib, imiquimod, interferon alfa-2b, ipilimumab, irinotecan,ixabepilone, ixazomib, lanreotide, lapatinib, lenalidomide, lenvatinib,letrozole, leucovorin, leuprolide, lomustine, mechlorethamine, megestrolacetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin C,mitoxantrone, necitumumab, nelarabine, netupitant, nilotinib,nilutamide, nivolumab, obinutuzumab, ofatumumab, olaparib, omacetaxinemepesuccinate, osimertinib, oxaliplatin, ozogamicin, paclitaxel,palbociclib, palifermin, pamidronate, panitumumab, panobinostat,pazopanib, pegaspargase, peginterferon alfa-2b, pembrolizumab,pemetrexed, pertuzumab, plerixafor, pomalidomide, ponatinib,pralatrexate, prednisone, procarbazine, propranolol, radium 223dichloride, raloxifene, ramucirumab, rasburicase, regorafenib,rituximab, rolapitant, romidepsin, romiplostim, ruxolitinib, siltuximab,sipuleucel-t, sonidegib, sorafenib, sunitinib, talimogene laherparepvec,tamoxifen, temozolomide, temsirolimus, thalidomide, thioguanine,thiotepa, tipiracil, topotecan, toremifene, toremifene, tositumomab,trabectedin, trametinib, trastuzumab, tretinoin, trifluridine, uridinetriacetate, vandetanib, vemurafenib, venetoclax, vinblastine,vincristine, vinorelbine, vismodegib, vorinostat, ziv-aflibercept,zoledronic acid, and pharmaceutically acceptable salts thereof. In someembodiments, the second therapeutic agent is one or more of rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone.

In some aspects, administration of a GSK-3β inhibitor in combinationwith a second therapeutic agent reduces the amount of the secondtherapeutic agent needed to produce a given therapeutic effect. That is,in some embodiments a therapeutically effective treatment comprisesadministering a GSK-3β inhibitor in combination with a sub-therapeuticamount (i.e., an amount that is ineffective when administered as thesole therapeutic agent) of a second therapeutic agent. In someembodiments, a GSK-3β inhibitor is administered in combination with asub-therapeutic amount of an apoptosis modulator. In some embodiments,9-ING-41 is administered in combination with a sub-therapeutic amount ofa Bcl-2 inhibitor. In some embodiments, 9-ING-41 is administered incombination with a sub-therapeutic amount of venetoclax, ABT-737, ornavitoclax. In some embodiments, 9-ING-41 is administered in combinationwith a sub-therapeutic amount of Venetoclax.

In other embodiments, tideglusib is administered in combination with asub-therapeutic amount of a Bcl-2 inhibitor. In some embodiments,tideglusib is administered in combination with a sub-therapeutic amountof venetoclax, ABT-737, or navitoclax. In some embodiments, tideglusibis administered in combination with a sub-therapeutic amount ofVenetoclax.

In yet other embodiments, LY2090314 is administered in combination witha sub-therapeutic amount of a Bcl-2 inhibitor. In some embodiments,LY2090314 is administered in combination with a sub-therapeutic amountof venetoclax, ABT-737, or navitoclax. In some embodiments, LY2090314 isadministered in combination with a sub-therapeutic amount of Venetoclax.

In other embodiments, a GSK-3β inhibitor is administered in combinationwith a sub-therapeutic amount of a CDK modulator. In other embodiments,a GSK-3β inhibitor is administered in combination with a sub-therapeuticamount of a CDK9 inhibitor. In other embodiments, a GSK-3β inhibitor isadministered in combination with a sub-therapeutic amount of a CDK9inhibitor. In some embodiments, 9-ING-41 is administered in combinationwith a sub-therapeutic amount of a CDK9 inhibitor. In some embodiments,9-ING-41 is administered in combination with a sub-therapeutic amount ofBAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887. In someembodiments, 9-ING-41 is administered in combination with asub-therapeutic amount of BAY-1143572.

In some embodiments, tideglusib is administered in combination with asub-therapeutic amount of a CDK9 inhibitor. In some embodiments,tideglusib is administered in combination with a sub-therapeutic amountof BAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887. In someembodiments, tideglusib is administered in combination with asub-therapeutic amount of BAY-1143572.

In some embodiments, LY2090314 is administered in combination with asub-therapeutic amount of a CDK9 inhibitor. In some embodiments,LY2090314 is administered in combination with a sub-therapeutic amountof BAY-1143572, LDC000067, Dinaciclib (SCH727965), SNS-032 (BMS-387032),AT7519, P276-00, AZD5438, PHA-767491, or PHA-793887. In someembodiments, LY2090314 is administered in combination with asub-therapeutic amount of BAY-1143572.

In some aspects, a GSK-3β inhibitor is administered in combination witha sub-therapeutic amount of a modulator of the MTOR/AKT/PI3 pathway. Insome aspects, a GSK-3β inhibitor is administered in combination with asub-therapeutic amount of a PI3K inhibitor. In some embodiments,9-ING-41 is administered in combination with a sub-therapeutic amount ofa PI3K inhibitor. In other embodiments, 9-ING-41 is administered incombination with a sub-therapeutic amount of copanlisib or idelalisib.In some embodiments, 9-ING-41 is administered in combination with asub-therapeutic amount of copanlisib. In yet other embodiments, 9-ING-41is administered in combination with a sub-therapeutic amount ofidelalisib.

In some embodiments, tideglusib is administered in combination with asub-therapeutic amount of a PI3K inhibitor. In other embodiments,tideglusib is administered in combination with a sub-therapeutic amountof copanlisib or idelalisib. In some embodiments, tideglusib isadministered in combination with a sub-therapeutic amount of copanlisib.In yet other embodiments, tideglusib is administered in combination witha sub-therapeutic amount of idelalisib.

In some embodiments, LY2090314 is administered in combination with asub-therapeutic amount of a PI3K inhibitor. In other embodiments,LY2090314 is administered in combination with a sub-therapeutic amountof copanlisib or idelalisib. In some embodiments, LY2090314 isadministered in combination with a sub-therapeutic amount of copanlisib.In yet other embodiments, LY2090314 is administered in combination witha sub-therapeutic amount of idelalisib.

The GSK-3β inhibitor may be administered in a pharmaceutical compositioncomprising the GSK-3β inhibitor and at least one pharmaceuticallyacceptable carrier or excipient. In some embodiments, 9-ING-41 may beadministered in a pharmaceutical composition comprising 9-ING-41 and atleast one pharmaceutically acceptable carrier or excipient. In otherembodiments, tideglusib may be administered in a pharmaceuticalcomposition comprising tideglusib and at least one pharmaceuticallyacceptable carrier or excipient. In other embodiments, LY2090314 may beadministered in a pharmaceutical composition comprising LY2090314 and atleast one pharmaceutically acceptable carrier or excipient. Similarly,the second therapeutic agent may be administered in a pharmaceuticalcomposition comprising the second therapeutic agent and at least onepharmaceutically acceptable carrier or excipient. Pharmaceuticallyacceptable carriers and excipients are known in the art. See, e.g.,Remington's Pharmaceutical Sciences, 18^(th) Edition, Mack PublishingCompany (1990).

In some aspects, the GSK-3β inhibitor and the second therapeutic agentmay be administered together in a single pharmaceutical composition.Thus, in some aspects, the present disclosure is directed topharmaceutical compositions comprising a GSK-3β inhibitor and a secondtherapeutic agent and at least one pharmaceutically acceptable carrieror excipient. In some embodiments, the pharmaceutical compositioncomprises a GSK-3β inhibitor; one or more of an apoptosis modulator, aCDK modulator, or a mTOR/AKT/PI3K modulator; and a pharmaceuticallyacceptable carrier or excipient. In some embodiments, the pharmaceuticalcomposition comprises one or more of 9-ING-41, tideglusib, or LY2090314;one or more of an apoptosis modulator, a CDK modulator, or amTOR/AKT/PI3K modulator; and a pharmaceutically acceptable carrier orexcipient. In some embodiments, the pharmaceutical composition comprisesone or more of 9-ING-41, tideglusib, or LY2090314; one or more ofVenetoclax, BAY-1143572, or idelalisib; and a pharmaceuticallyacceptable carrier or excipient. In some embodiments, the pharmaceuticalcomposition comprises one or more of 9-ING-41, tideglusib, or LY2090314;Venetoclax; and a pharmaceutically acceptable carrier or excipient. Inother embodiments, the pharmaceutical composition comprises one or moreof 9-ING-41, tideglusib, or LY2090314; BAY-1143572; and apharmaceutically acceptable carrier or excipient. In yet otherembodiments, the pharmaceutical composition comprises one or more of9-ING-41, tideglusib, or LY2090314; idelalisib; and a pharmaceuticallyacceptable carrier or excipient.

Representative methods of administration of the pharmaceuticalcompositions and combination therapies also are provided. Variousembodiments of the present invention further relate to methods ofadministering a pharmaceutical composition or combination therapy to ahuman patient for the treatment of lymphoma. The methods may compriseadministering a pharmaceutical composition or combination therapy bygenerally accepted routes of administration (e.g., oral, intravenous,subcutaneous, parenteral, inhalation, topical, etc.). In some instances,a pharmaceutical composition or combination therapy may be administeredorally, intravenously and/or subcutaneously. The administration may bemade using any suitable dosing regimen. Suitable dosing regimen areknown to persons of ordinary skill in the art.

In certain embodiments of the present invention, a pharmaceuticalcomposition or combination therapy may be administered to a humanpatient once daily. In other embodiments, a pharmaceutical compositionor combination therapy may be administered to a human patient twicedaily. In some embodiments, a pharmaceutical composition or combinationtherapy may be administered to human patients between meals.

In other aspects of the present disclosure, the GSK-3β inhibitor may beadministered in combination with another, non-chemotherapeutic,anti-cancer treatment. In some embodiments, the GSK-3β inhibitor isadministered in combination with radiation therapy. Thus, in someembodiments, the method of treating malignant lymphoproliferativedisorders comprises administering 9-ING-41 in combination with radiationtherapy. In some embodiments, the method of treating malignantlymphoproliferative disorders comprises administering tideglusib incombination with radiation therapy. In yet other embodiments, the methodof treating malignant lymphoproliferative disorders comprisesadministering LY2090314 in combination with radiation therapy.

EXAMPLES

The following examples further illustrate certain aspects of thedisclosure, and is not intended to limit the scope of the presentdisclosure in any way.

Materials—Examples 1-5

Daudi (Burkitt) and SUDHL-4 (germinal center (GC) diffuse large B-celllymphoma (DLBCL)) cell lines were purchased from American type; culture;collection. (ATCC). KPUM-UH1 (double hit DLBCL) cells were obtained fromJunya Kuroda, Kyoto Prefectural University of Medicine, Kyoto, Japan.All cell lines were cultured aseptically and maintained in awater-jacketed incubator (Thermo-Forma) at 37° C. with 5% CO2 and fedwith RPMI-1640 (Corning) containing 0.3 g/mL glutamine, 10% FBS (Sigma),and antibiotic and antimycotic reagent (Gemini Bioproducts; finalconcentrations—100 units/mL penicillin G, 100 μg/mL streptomycinsulfate, and 250 ng/mL amphotericin B). Karpas 422 (GC-DLBCL) cells waspurchased from Sigma. TMD8 (Activated B-Cell (ABC) DLBCL) cells wereobtained from the lab of Dr. Louis Stoudt from the NCI and weremaintained as above, but with 20% FBS. All cell numbers for assays werequantified using a TC20 automated cell counter (BioRad). All lymphomacell lines tested express active GSK-3β.

Venetoclax and Idelalisib were purchased from Selleck Chemicals.BAY-1143572 was purchased from Active Biochem. All drugs werere-suspended in DMSO. DMSO containing no drugs was used as ano-treatment control.

Example 1—Viability and Proliferation Assays (MTS Assay)

Cell viability at day 3 and proliferation over the course of 7 days weremeasured after treating cells with varying concentrations of 9-ING-41using the Promega CellTiter 96® AQueous One Solution Cell ProliferationAssay reagent (MTS) in accordance with the manufacturer's instructions.At the end of the treatment, 20 μL of reagent was added per well in a96-well plate and incubated for 2-4 h at 37° C. The absorbance at 490 nm(A490) was determined using a Powerwave XS plate reader (Biotek).

All lymphoma cell lines used in this study express active GSK-3β.SUDHL-4, KPUM-UH1, Karpas 422, or TMD8 lymphoma cells were plated, andcell numbers on days 1, 3, 5 and 7 were measured using the MTS assay.See FIG. 1. Cell viability on day 3 (FIG. 1A) was reduced 40-70%(p<0.05) upon 1 μM 9-ING-41 treatment, with SUDHL-4 and KPUM-UH showingthe highest reduction in cell viability. Upon exposure to 1 μM 9-ING-41,all lymphoma cell lines underwent growth arrest (FIG. 1B-IE) withproliferation of less than 30% on day 7, relative to control (p<0.05).Cell viability of lymphoma cells with varying concentrations of 9-ING-41(0.1 μM, 0.5 μM, 1 μM, 5 μM and 10 μM) was also tested, and a reductionin viability was seen at concentrations of 9-ING-41 that were 0.5 uM orhigher.

Example 2—EnzChek® Caspase 3 Assay

An EnzChek caspase 3 assay (Thermo Fisher Scientific) was performed inaccordance with the manufacturer's instructions. 100,000 cells wereplated in a 12-well plate and treated with varying concentrations of9-ING-41 for 24 hours in duplicate. At the end of treatment, cells werecentrifuged at 200 rcf for 5 minutes and washed once with 1×PBS andlysed in 50 μL of 1× lysis buffer provided by the kit. For efficientlysis, cells were subjected to a single freeze-thaw cycle. The lysedcells were centrifuged again to remove cell debris, and the supernatantwas used in the assay. 50 μL of 2× substrate working solution containingZ-DEVD-R110 substrate was added to the cell lysate, with a subsequentincubation at room temperature for 45 minutes. The rhodamine 110-derivedsubstrate (Z-DEVD-R110) used in this assay is a non-fluorescent bisamidecompound that, upon enzymatic cleavage via active caspase 3 and maybecaspase 7 in the cell lysates, is converted in a two-step process to thefluorescent monoamide and then to the even more fluorescent R110product. Both of these products were then measured using a Bioteksynergy 2 fluorescent plate reader at corresponding wavelengths(excitation 496 nm/emission 520 nm). The fluorescent reading wasnormalized to the amount of protein in the cell lysate as determined viastandard BCA assay (Pierce Thermo Fisher Scientific). Relativefluorescence was calculated after setting the no-treatment control to 1.

The ENZCHEK Caspase 3 assay revealed an increase in observed Caspase 3/7activity when lymphoma cells were treated with 0.5 μM or higherconcentrations of 9-ING-41. Pharmacokinetic studies in Xenograft micesuggest that 20 mg/kg intravenous administration can provide around 8 μM9-ING-41 concentration in plasma and around 40 μM 9-ING-41 in the brainwithin 30 minutes. Ugolkov A, Qiang W, Bondarenko G, Procissi D, GaisinaI, James C D, Chandler J, Kozikowski A, Gunosewoyo H, O'Halloran T,Raizer J, Mazar A P. Combination Treatment with the GSK-3 Inhibitor9-ING-41 and CCNU Cures Orthotopic Chemoresistant Glioblastoma inPatient-Derived Xenograft Models. Transl Oncol. 2017; 10:669-78.https://doi.org/10.1016/j.tranon.2017.06.003.

The MTS and ENZCHEK Caspase 3 assay data show that 9-ING-41 inhibitsproliferation of lymphoma cell lines as a single agent and reducesviability of lymphoma cells. Without intending to be bound by theory,these results suggest that GSK-3β targeting leads to decreasedproliferation and viability of aggressive B-cell lymphoma cell lines byinducing variable effects on pro-survival signals and DNA damageresponse, ultimately leading to apoptosis. These effects wereindependent of cell of origin in the DLBCL cell lines.

The activity of 9-ING-41 in the DHL cell line KPUM-UH1, a typicallychemotherapy-resistant cell line, is of particular interest. Withoutintending to be bound by theory, Luminex analysis (described below)suggests that 9-ING-41 exerts effects in the KPUM-UH1 cell line throughdown-regulation of c-MYC signaling and induction of apoptosis throughreduction of survivin. This down-regulation of surviving does not appearto be associated with, or driven by, changes in NF-κB in this cell line.This is in contrast to what has been described in acute lymphoblasticleukemia (ALL) where GSK-3β suppression sensitizes ALL cells toNF-κB-mediated apoptosis via survivin effect. See Hu Y, Gu X, Li R, LuoQ, Xu Y. Glycogen synthase kinase-3beta inhibition induces nuclearfactor-kappaB-mediated apoptosis in pediatric acute lymphocyte leukemiacells. J Exp Clin Cancer Res. 2010; 29:154.https://doi.org/10.1186/1756-9966-29-154.

Example 3—Western Blot Analysis

c-MYC levels were analyzed in KPUM-UH1 cells via western blot for9-ING-41 alone and the combination of 9-ING-41 with either Venetoclax orBAY-1143572. Around 10 million cells were spun down at 200 rcf for 5minutes and rinsed once with PBS before lysing in 50 μl of MilliporeMilliplex MAP lysis buffer supplemented with protease and phosphataseinhibitors (Roche). Protein denatured in 4× sample buffer supplementedwith β-mercaptoethanol (Bio-Rad) was loaded per well. Bio-Rad stain-freeCriterion 4-20% precast gels were used. After running the gel at 140volts for 90 minutes, Bio-Rad gel imager was used to activate thestain-free technology to visualize the total protein levels loaded inthe gel. A nitrocellulose turbo-transfer pack and system (Bio-Rad) wasthen used to transfer the proteins to the membrane, and 5% w/v dry milkin Tris-buffered saline-0.1% Tween 20 (TBS-T) was used to block themembranes for 1 hour. Membranes were then incubated with the primaryantibody diluted in 5% BSA in TBS-T overnight. Membranes were thenrinsed in TBS-T 3 times (one 15 minute wash and two 5 minutes washes)and then incubated with the corresponding secondary antibody-conjugatedwith HRP for 1 hour and rinsed with TBS-T as before. After the finalwash, membranes were developed using a Pierce SuperSignal West Picochemiluminescence kit and visualized using the Bio-Rad imaging system.Antibodies used included: Rabbit anti-GSK-3D (Cell Signaling, Cat. No:12456), Rabbit anti-phospho-GSK-3β (Y216) (Abcam, Cat. No: ab75745),Rabbit anti-c-MYC (Cell Signaling, Cat. No: 5605), Rabbitanti-phospho-c-MYC (Ser 62) (Cell Signaling, Cat. No: 13748), Rabbitanti-phospho-c-MYC (Thr58) (Abcam, Cat. No: ab185655), and Mouseanti-β-Actin (Sigma, Cat. No: A5441) at a 1:1000 dilution. Anti-RabbitHRP and Anti-Mouse HRP secondary antibodies were purchased from CellSignaling and used at a 1:5000 dilution. When necessary, membranes werestripped using RESTORE PLUS Western blot stripping buffer (Pierce) for10 mins and washed with TBS-T several times and re-blocked and re-probedas before. Quantification of the band intensities were performed usingImage J software (NIH). These experiments in the double-hit lymphomacell line, KPUM-UH1, suggest that phospho-c-MYC is modified with9-ING-41 treatment.

Example 4—Luminex Analysis

Signaling changes in NF-κB [MILLIPLEXMAP NF-κB Signaling Magnetic BeadKit 6-plex Kit, EMD Millipore, analytes: c-MYC, FADD (Ser194), IκBα(Ser32), IKKα/β (Ser177/Ser181), NF-κB (Ser536), TNFR1], DNA damage[MILLIPLEX MAP DNA Damage/Genotoxicity Magnetic Bead Panel, EMDMillipore, analytes: ATR (total), Chk1 (Ser345), Chk2 (Thr68), H2A.X(Ser139), MDM2 (total), p21 (Total), p53 (Ser15)], and apoptoticpathways [Bio-plex pro RBM apoptosis panel 2 and 3, Bio-Rad, analytes:Bad, Bax/Bcl-2 dimer, Bcl-xL, Bim, Mcl-1, active caspase 3, Bcl-xL/Bakdimer, Mcl-1/Bak dimer, survivin] associated with 1 μM 9-ING-41treatment for 48 hours as compared to no-treatment controls weredetermined using Luminex multiplex technology with a FLEXMAP 3Dinstrument, as per manufacturer instructions. Cells were lysed inMILLIPLEX MAP Lysis buffer supplemented with protease inhibitor cocktail(Sigma) and phosphatase inhibitor cocktails 2 and 3 (Sigma) and, afterBCA protein determination, 15 μg of protein was added to each well. Allsamples were run in duplicate and changes in MFI or absolute quantitybetween 9-ING-41-treated cells and nontreated control were analyzed, andan unpaired t-test was performed to determine statistical significance.

Analysis of NF-κB signaling showed a significant reduction in totalc-MYC levels in Karpas 422 and TMD8 cell lines but only trends forreduction in this protein in the remaining cell lines. DNA damagesignaling, via evaluation of p-H2A.X (Ser139), was found to be increasedfor SUDHL-4 and Karpas 422 cell lines (both p<0.05). In addition, asignificant increase in phospho-p53 (Ser15) upon 9-ING-41 treatment wasobserved in SUDHL-4 and TMD8 cells. Apoptosis signaling pathway analysisrevealed a significant reduction (˜2-fold, p<0.05) in survivin and anincrease in active caspase 3 in all lymphoma cell lines except TMD8.With the exception of KPUM-UH1, all lymphoma cell lines showedsignificant reductions (˜2-fold, p<0.05) in Mcl-1/Bak dimers, whileBcl-xl/Bak dimer expression was significantly (˜1.5-fold, p<0.05)reduced in all cell lines except TMD8.

Example 5—Dose Response of Combinations

Daudi, SUDHL-4, KPUM-UH1, Karpas 422, or TMD8 cells were treatedsimultaneously with a series of concentrations of 9-ING-41 (0 μM, 0.05μM, 0.5 μM) and either Venetoclax (0 nM, 0.5 nM, 5 nM, 50 nM, 500 nM,5000 nM), BAY-1143572 (0 μM 0.005 μM, 0.05 μM 0.5 μM, 5 μM, 50 μM), orIdelalisib (0 μM, 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, 50 μM). See FIG. 2.Viability at day 3 using an MTS assay was determined as described above.The background absorbance was subtracted from the A490 of the samplesand the A490 of the vehicle/no-treatment control was set to 1, and therelative A490s of the rest of the samples were calculated. The IC50 wascalculated as the concentration of the drug at which A490 reached 0.5.Additive effects were calculated as a fold change in the IC50 of a novelagent when combined with 0.5 μM of 9-ING-41.

As shown in FIG. 3, combination therapy using 9-ING-41 and a secondtherapeutic agent can reduce the amount of the second therapeutic agentrequired to produce a given therapeutic effect. As shown, combinationtreatment of the SUDHL-4 cell line with 0.5 μM 9-ING-41 showed 8-foldreduction in the IC50 value of Venetoclax. Similarly, combinationtreatment of the KPUMUH1 cell line with 0.5 μM 9-ING-41 showed 2-foldreduction in the IC50 value of Venetoclax. Combination treatment of theSUDHL-4 cell line using 0.5 μM 9-ING-41 showed an 8-fold reduction inthe IC50 value of BAY-1143572. Combination treatment using 9-ING-41 didnot significantly changes in the IC50 values of Idelalisib in thestudied cell lines.

Materials and Methods—Examples 6-11

Lymphoma tissue samples were obtained from patients after writteninformed consent. This Lymphoma SPORE biospecimens protocol was approvedby the Mayo Clinic Institutional Review Board in accordance with theDeclaration of Helsinki. All primary patient samples were biopsy tissuesfrom spleen or lymph nodes. Fresh tissue samples were gently dissociatedinto cell suspension and subjected to Ficoll-Paque density gradientcentrifugation. Primary lymphoma cells were then used directly forproliferation assay, or stored in −80° C. for Western analysis laterafter lymphoma diagnosis confirmation.

All lymphoma cell lines used in this study were purchased from ATCC(Manassas, Va.) or DSMZ (Braunschweig, Germany). DLBCL (Diffuse LargeB-Cell Lymphoma) lines were cultured in IMDM medium supplemented with10% human serum (Sigma-Aldrich); TCL (T-cell non-Hodgkin lymphoma) andMCL (Mantle Cell Lymphoma) lines were maintained in RPMI-1640 mediumsupplemented with 10% fetal calf serum. The Jeko cell line used forxenograft modeling in mice were stably expressed with Firefly luciferase(Fluc) through lentiviral transduction. Cell lines are periodicallychecked for the absence of mycoplasma infection, and authenticated byeither home brew SNP-based PCR method or short tandem repeat profilingthrough ATCC.

Antibodies, and Other Reagents

Common agents were from Sigma-Aldrich. Antibodies for immunoblotting,including anti-human GSK3α (Cat #4337), GSK3β (Cat #12456),phospho-GSK3α-S21/GSK3β-S9 (Cat #9327) were purchased from CellSignaling. Mouse monoclonal anti-GSK3β (clone 7/GSK3β, Cat #610201, BDBiosciences), anti-α-tubulin (clone MD1A, Cat #T9026) and rabbitanti-pericentrin (Cat #ab4448) antibodies used for immunofluorescencewere from Sigma and Abcam, respectively. Alexa 488 or Alexa 565conjugated secondary antibodies were products of Life Technologies.

Apoptosis Assay

Cells were seeded at 5×10⁵ cells/well in 24 well plates, and incubatedwith 9-ING-41 at indicated concentrations for 48 hours. The cells werethen stained with FITC conjugated Annexin V (Life Technologies) andPropidium Iodide followed by analysis on a BD FACS Calibur flowcytometer.

DNA Cell Cycle

Cells were fixed and permeabilized with cold ethanol, treated withRNase, and stained with propidium iodide (Sigma). Stained cells were runon a BD FACS Calibur using CELLQuest PRO Software (Becton Dickinson).Data were analyzed with FlowJo v.X software (Tree Star Inc).

Proliferation Assay

Cells were seeded at 1×10⁴ cells/well in 96 well plates, and incubatedwith 9-ING-41 at indicated concentrations for 48 hours followed bypulsing with tritium labeled thymidine for overnight before beinganalyzed for thymidine uptake.

Western Immunoblotting

Western analysis was performed as previously described and developed ona LI-COR Odyssey CLX imager using LI-COR reagent system.

Quantitative PCR

Total mRNAs from lymphoma cell lines were isolated using an RNAeasy kit(Qiagen) and cDNAs synthesized with a Superscript III cDNA synthesis kit(Life technology). The qPCR were then performed on an ABI 7500 Real-TimePCR Systems (Applied Biosystems) using RT² SYBR Green ROX qPCR Mastermix(Qiagen).

Drug IC50 Calculation

The IC_(50S) of 9-ING-41 on cell survival and proliferation wascalculated using an on-line IC50 calculation tool(https://www.aatbio.com/tools/ic50-calculator/).

Immunohistochemistry Staining

Immunohistochemistry (IHC) on 5 μm thick paraffin sections was performedaccording to standard protocol. Briefly, tissue sections on slides weredeparaffinized with xylene, rehydrated with series of alcohol, followedby antigen retrieval in citrate buffer (pH 6.0). The resulting slideswere endogenous peroxidase quenched with 30% hydrogen peroxide. Slideswere then incubated with anti-GSK3b antibody (BD, 1:150) at roomtemperature for 2 hours, washed three times with Tris-buffered saline (5min each) and incubated with biotinylated anti-mouse secondary antibody(1:200) for 1 h at room temperature. After treating the slides withHRP-conjugated ABC complex (Vectastain, Vector Laboratories) for 1 h atroom temperature, color was developed with 3,39-diamino Benzidine (DAB,Vector Laboratories) counterstained with methylene blue, mounted withDPX and examined and imaged on a Nikon Eclipse Ti microscope. negativecontrols were performed on patient samples without addition of primaryantibody.

Immunofluorescence Staining for Centrosome and Spindle Localization ofGSK3β

For the coimmunostaining of GSK3β and pericentrin at centrosomes (FIG.8C-8J), we used an incomplete fixation method by fixing the cells oncytospin slides with 3% paraformaldehyde in PBS for 5 min at roomtemperature. The cells were then permeabilized with 0.2% Triton X-100 inPBS for 10 min followed by blocking with 5% BSA in PBS for 1 hour andimmunostained with mouse anti-GSK3β mAb and rabbit anti-pericentrinovernight at 4° C. The cells were then washed and further stained withfluorochrome conjugated secondary antibodies.

All other immunostainings of GSK3β and α-Tubulin on cytospinpreparations of lymphoma cells were fixed with 4% paraformaldehyde inPBS for 15 min at room temperature followed by permeabilization and thesame staining steps as above. The cells were analyzed and imaged on aconventional Zeiss microscope or a Zeiss LSR 780 confocal microscope.

Example 6. GSK3α and GSK3 are Overexpressed in Lymphoma Cells

The expression status of GSK3α and GSK3β in purified human normal Band Tcells and DLBCL, MCL, and TCL lymphoma cell lines was examined. ByRT-qPCR, it was found that most lymphoma lines showed higher butvariable levels of GSK3α and GSK3β mRNAs compared to the lowerexpression in normal lymphocytes (FIG. 4A), indicating that thetranscription of GSK3 is enhanced in most of lymphoma cell lines. GSK3protein expression by was analyzed by Western blotting, showing thatmost lymphoma lines (except Ly-19) showed strong expression of GSK3αprotein compared to weak expression in B and T lymphocytes (FIG. 4B, ingreen). Similarly, GSK3β protein was also strongly expressed in alllymphoma cell lines but very weakly expressed (visible after longexposure; not shown) in normal lymphocytes (FIG. 4B, in red). These dataindicate that GSK3 proteins are overexpressed in most B- and T-lymphomacell lines.

By Western blotting, both GSK3α and GSK3β proteins were shown to bevariably phosphorylated across our lymphoma line panel similar to normallymphocytes.

Example 7. GSK3α and GSK3 Fare Functionally Important in Lymphoma Cells

Given that both GSK3α and GSK3β are overexpressed in lymphoma cells, andthat both enzymes are implicated in multiple signaling pathways criticalfor cell functions, whether GSK3α and GSK3β are functionally supportingthe survival and proliferation of lymphoma cells was examined. Treatmentof TCL and MCL lines with low doses of 9-ING-41 for 48 hours inducedapoptosis (FIG. 5A); the DLBCL lines required higher concentrations(FIG. 5B). In contrast, no significant apoptosis in purified normalunstimulated T lymphocytes or peripheral blood mononuclear cells wasdetected even at a concentration of 10.0 μM of 9-ING-41. The inhibitoryconcentrations of 9-ING-41 at half of the maximal effect (IC₅₀) on cellsurvival of various lymphoma cell lines was calculated (Table 1).

TABLE 1 9-ING-41 inhibitory concentrations at 50% of the maximum (IC₅₀)on cell survival and cell proliferation in various lymphoma cell lines.IC₅₀ (μM) on IC₅₀ (μM) on Lymphoma line Cell survival Cell proliferationDLBCL OCI-LY1 3.05 0.69 OCI-LY19 2.21 1.96 OCI-Ly3 3.76 1.03 SU-DHL61.58 0.84 1.58 0.84 MCL Granta-519 0.77 0.38 Jeko 1.28 0.94 Mino 0.550.72 TCL Karpas-299 3.34 0.26 MyLa 0.69 0.19 SeAx 1.60 0.38

9-ING-41 can specifically induce lymphoma cell apoptosis withoutaffecting normal lymphocytes. The thymidine incorporation assay in thepresence or absence of 9-ING-41 was performed to test the role of GSK3in lymphoma cell proliferation. The proliferation rate of all TCL andMCL lines was profoundly inhibited in the presence of 9-ING-41concentrations as low as 1.0 μM. The DLBCL lines required slightlyhigher concentrations. The IC₅₀ of 9-ING-41 on cell proliferation forvarious lymphoma cell lines was calculated (Table 1). These dataindicate that GSK3 activity is important for the proliferation andsurvival of lymphoma cells.

CRISPR/CAS9 knockout technique was used to genetically delete GSK3A andGSK3B genes.

Guide RNAs (gRNAs) for targeting the 1st coding exons of both GSK3α andGSK3β genes were designed using a web tool (http://crispr.mit.edu/). ThegRNA sequences (GSK3α: GACAGATGCCTTTCCGCCGC; GSK3β:CGGCTTGCAGCTCTCCGCAA), were cloned into the px458 vector (Addgene)carrying a co-expressing GFP. The constructs were nucleofected intolymphoma cells using a nucleofection kit (Lonza, Basel, Switzerland).Thirty-six hours post nucleofection, GFP expressing single cells weresorted into 96 well plates at 1 cell/well on an Aria II FACS sorter.After the expansion of single cell subclones in culture for two weeks,each subclone was genotyped by PCR and Sanger's DNA sequencing.

After transient expression of the construct carrying CAS9-T2A-GFP andgRNA specific GSK3A or GSK3B genes exon 1 sequences, GFP expressingsingle cells were sorted into 96-well plate by flow sorting. After 2-3weeks in culture, single cell subclones carrying unique modification inGSK3A, GSK3B, or both genes were genotyped for the gene deletion andwestern blot verified for the protein depletion. As summarized in Table2, several GSK3A null knockout subclones were readily obtained from all5 cell lines tested, and GSK3B null subclones were also obtained fromLy-1 cell lines. However, after analyzing 24-36 single cell subclonesfrom Ly-19, Jeko, Mino, and Karpas 299 cell lines, no knockout subcloneswere detected; i.e., all survived clones were either wildtype or carriedheterozygous mutations suggesting GSK3B null cells from those cell lineslikely died during culture. Given that CRISPR/CAS9 is a highly efficientapproach for biallelic deletion of GSK3B gene in Ly-1 cells (19/24, 79%)but no GSK3B null clones in any other lymphoma lines tested (0/24, 0/24,0/36, 0/26, 0%), these data support the conclusion that GSK3B isnecessary for the survival of lymphoma cells in these cell lines. UsingshRNA knockdown similar results were also observed, showing that GSK3Bknockdown is lethal in several lymphoma lines except Ly-1.

TABLE 2 Effect of GSK3α and/or GSK3β knockout using CRISPR/Cas9 approachon the survival of various lymphoma cell lines Null knockoutclones/total clones screened Lymphoma line GSK3α GSK3β GSK3α/β OCI-LY19/17 19/24  6/13 OCI-LY19 20/28  0/24 not done Jeko 7/12 0/24 not doneMino 5/12 0/36 not done Karpas299 12/20  0/26 not done

Example 7. GSK3 Inhibition Blocks G2/M Progression in Lymphoma Cells

The effect of 9-ING-41 on lymphoma cell cycle kinetics was examined.After 9-ING-41 treatment, cell cycle blockage at G2M after as little as24 hours of treatment in all lines tested (FIG. 6A), indicating thatGSK3 activity is required for successful progression of mitosis. Tofurther determine whether this G2/M arrest specifically resulted fromGSK3β inhibition, the cell cycle profile of parental (wildtype), GSK3A,GSK3B, and GSK3A/B knockout Ly-1 subclones was examined. In the absenceof treatment, both parental wildtype and GSK3A-null Ly-1 cells showednormal cell cycle profiles, while GSK3B and GSK3A/B knockout subclonesexhibited increased cells in G2/M (FIG. 6B). In addition, GSK3A1B doubleknockout subclones also showed increased levels of polyploid (>4N)cells, possibly due to defective mitosis. These cell cycle abnormalitiesof GSK3B-null and GSK3A-B-null Ly-1 cells are subtle and have littleimpact on the survival of the Ly-1 progeny, likely through Ly-1 cellline specific compensatory mechanisms. 9-ING-41 treatment phenocopiesthe effect of GSK3B single or GSK3A-B double deletion on cell cycleprogression indicates that GSK3B is critical for lymphoma cell cycleG2/M progression, and that 9-ING-41 is a potent cell cycle blockingagent for lymphoma cells.

Example 8. GSK3 Inhibition Arrests Lymphoma Cells at the Prophase Stageof Mitosis

Although cells arrested in G2/M appear as a single DNA content (4N) peakon a flow cytometry histogram (FIG. 6A), there are actually at least 5sequential steps (M1-M5) in G2/M critical to successful cell division.These include prophase (M1, chromosome condensation, mitotic spindleformation starts), prometaphase (M2, nuclear membrane breakdown,centrosome polarization), metaphase (M3, chromosome pairs align middleplane), anaphase (M4, daughter chromatids separate), telophase (M5,reformation of daughter nuclei), and the final cytokinesis (separationof two daughter cells). Each of these discrete steps has a uniqueidentifiable nuclear morphology on Wright's stained cells (depicted inFIG. 7A). The morphology of untreated and 9-ING-41 treated Jeko cells todetermine at what stage they become arrested was examined. As shown inFIG. 7B (left panel), all M1-M5 mitotic steps were readily identified inuntreated mitotic Jeko cells; however, in 9-ING-41 treated cells (rightpanel) a large fraction of cells showed the morphology of condensedchromosomes and reduced cytoplasmic staining resembling prophase (M1)cells without identifiable cells with M2-M5 morphology. By differentialcounting of 100 mitotic cells, all stages (M1-M5) of mitotic cells werefound readily identifiable in untreated cells; only prophase (M1) cellswere accounted for in 9-ING-41 treatment mitotic cells (FIG. 7C).Similar results were observed in other lymphoma lines including DHL-6,Ly-3, Mino and Karpas 299. These observations support the conclusionthat GSK3 activity is necessary for the progression of mitotic prophase.

Example 9. GSK3β is Localized to Centrosomes and Mitotic Spindles ofLymphoma Cells

The involvement of GSK3β in two key prophase events, centrosomepolarization and mitotic spindle formation was examined. The subcellularlocalization of GSK3β protein in interphase Jeko or Ly-1 cells wasexamined by immunofluorescence staining. During interphase, GSK3β isprominently localized in the nucleus and centrosome-like pair dots inthe cytoplasm of Jeko cells (FIG. 8A-B). To further demonstrate thatthose cytoplasmic pair dots were indeed centrosomes, wt Ly-1 cells werefirst costained for GSK3β protein and the centrosome marker pericentrinusing an incomplete fixation protocol and it was found that thecytoplasmic GSK3 dots were perfectly colocalized with pericentrin (FIG.8D-8F), indicating these GSK3β bright dots (in green, FIGS. 8A-C & 8F)are indeed centrosomes. It was further demonstrated that the anti-GSK3βantibody staining was specific to GSK3 protein by showing its absence inGSK3B-null Ly1 cells (FIG. 8G-J). Therefore, we conclude that GSK3β islocalized to centrosomes and the nucleus in interphase cells.

To determine the intracellular localization of GSK3β in mitotic cells,GSK3β localization in normal mitotic Jeko cells was analyzed. GSK3βstaining (in green, FIG. 8K-L) exhibited a “firework-like” pattern withpolarized centrosomes in the middle and mitotic spindles or microtubulesextending outwards. The staining is specific to GSK3β since suchstaining is absent in GSK3B deficient mitotic cells (FIG. 8O-P).α-Tubulin gives a staining pattern (using a separate stain formicrotubule marker α-tubulin) similar to that of GSK3β (in red, FIG.8M-N) suggesting that GSK3β is localized to microtubules during mitosis.These observations collectively indicate that GSK3β protein isspecifically localized to centrosomes and mitotic spindles duringmitosis. The localization of GSK3β in 9-ING-41 treated Jeko cells (FIG.8Q-R) was determined. A similar firework-like GSK3β staining patternswere observed in all prophase cells without any altered GSK3βlocalization. Taken together, these data indicate that GSK3β localizedto centrosomes and nucleus during interphase (FIG. 8A-B), and tocentrosomes and mitotic microtubules during mitosis (FIG. 8K-L).Treatment of 9-ING-41 did not alter the localization of GSK3β, noraffect centrosome polarization or microtubule formation; therefore,9-ING-41 is likely to inhibit microtubule function at a later steppreventing the progression of prophase.

Example 10. GSK3 Expression and Targeting in Primary Cells from LymphomaPatients

The expression of GSK3α and GSK3β proteins in primary lymphoma cellsfreshly isolated from patients with MCL, high grade B-cell lymphoma,follicular lymphoma grade 3B, DLBCL, or angioimmunoblastic TCL wereexamined. All 5 samples had stronger expression of GSK3α and GSK3βproteins compared to normal blood B-cell controls (FIG. 9A). Thesepatient cells were similarly responsive to the antiproliferative effectsof 9-NG-41 (FIG. 9B). Paraffin samples from patients of a differentcohort with various lymphoma types were probed for GSK3 proteinexpression by IHC. As shown in FIG. 9C, GSK3β overexpression in allsamples was found with variable intensity. RNA-Seq analysis on primaryDLBCL patient samples derived from a public database at Gene ExpressionProfile Interactive Analysis (http://gepia.cancerpku.cn), also showedincreased expression of GSK3α and GSK3β in lymphoma than normallymphocytes.

Recently published RNA-Seq data (Schmitz R, Wright G W, Huang D W, etal. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N Engl JMed. 2018; 378(15):1396-1407) on a cohort of 234 DLBCL patients withclinical survival data [median follow-up was 10.5 years (95% CI: 7.9—notreached)] was analyzed. A receiver operating characteristics (ROC) curveanalysis was performed to dichotomize the GSK3α and GSK3β expression toestablish the optimal cutoffs (10.5 for GSK3α, and 8.6 for GSK3β) forhigh and low expression grouping. The GSK3α high expression group(≥10.5, n=172) had an OS of 7.8 years (95% OS: 7.2-8.4) while to GSK3αlow expression group (<10.5, n=62) had a significantly (p=0.03) higherOS of 8.9 years (95% OS: 8.2-10.1). Similarly, GSK3β high expressiongroup (≥8.6, n=170) had an OS of 7.8 years (95% OS: 7.2-8.2) while toGSK3β low expression group (<8.6, n=62) had a significantly (p=0.0005)higher OS of 9.7 years (95% OS: 8.6-11.5). These results suggest thatoverexpression of GSK3α or GSK3β each correlates with poorer clinicaloutcome. In addition, the grouping data also show that the majority ofDLBCL patients are segregated in high expression group for either GSK3αor GSK3β further validating the conclusion that GSK3α and GSK3β aregenerally overexpressed in lymphoma.

Example 11. Targeting GSK3 in Mouse Xenografts of Human Lymphoma

An MCL xenograft mouse model was established by subcutaneously injectingNGS mice with Jeko cells expressing the firefly luciferase reporter geneFluc.

NSG (NOD.Cg-Prkdc_(scid)Il2rg_(tm1Wjl)/SzJ) mice used in theseexperiments were purchased from the Jackson Laboratory (Bar Harbor,Me.). Eight to ten mice of same sex at 8 weeks of age weresubcutaneously injected with 5×10⁶Fluc expressing Jeko cells in theright flanks. Tumor engraftment was verified by imaging 4 days afterJeko cell inoculation. The tumor engrafted mice were randomly groupedinto control and treatment groups, then untreated or treated with9-ING-41 by IP injection as indicated. Tumor volumes were measured by anIVIS Imager (Xenogen, Alameda, Calif.) 20 minutes after IP injection of200 ul of 15 mg/ml D-leuciferin (GoldBio, St. Louis, Mo.) andanesthetized with 2.5% isoflurane. All imaging variables were keptconsistent for comparativeness. The experiment terminated when thelargest tumor met the size limit of the IACUC protocol.

In two independent experiments, eight and ten mice with engrafted tumorsverified by imaging (typically 4 days after tumor inoculation) randomlyserved as controls or received 9-ING-41 treatment 40 mg/kg every otherday IP (FIG. 10A). As shown in FIG. 10B, the control (untreated) groupmice had large tumors with marked luciferase activities by the day 17;however, the 9-ING-41 treated mice had smaller tumors with much lowerluciferase activity. These data demonstrated that 9-ING-41 hassingle-agent anti-tumor activity in a mouse model of MCL.

Example 12 Treatment of Diffuse Large B-Cell Lymphoma (DLBCL) Using9-ING-41

A human suffering from Diffuse large B-cell lymphoma is administered 1mg/kg per day of 9-ING-41 for six 21 day cycles. After treatment, thehuman's DLBCL is in remission.

Example 13 Treatment of Mantle Cell Lymphoma (MCL) Using 9-ING-41

A human suffering from Mantle Cell lymphoma is administered 3 mg/kg perday of 9-ING-41 for six 21 day cycles. After treatment, the human's MCLis in remission.

Example 14 Treatment of T-Cell Lymphoma (TCL) Using 9-ING-41

A human suffering from T-cell lymphoma (non-Hodgkin's) is administered 2mg/kg per day of 9-ING-41 for six 21 day cycles. After treatment, thehuman's TCL is in remission.

See Karmali, et al., GSK-3β inhibitor, 9-ING-41, reduces cell viabilityand halts proliferation of B-cell lymphoma cell lines as a single agentand in combination with novel agents, Oncotarget. 2017 Dec. 29; 8(70):114924-114934, which is incorporated by reference herein in itsentirety.

See Wu, et al., Targeting glycogen synthase kinase 3 for therapeuticbenefit in lymphoma, Blood. Published online May 17, 2019(http://www.bloodjournal.org); doi:10.1182/blood.2018874560, which isincorporated by reference herein in its entirety.

What is claimed:
 1. A method of treating a malignant lymphoproliferativedisorder in a patient in need thereof, comprising administering to saidpatient an effective amount of 9-ING-41.
 2. The method of claim 1,wherein the malignant lymphoproliferative disorder is a malignant B-celllymphoproliferative disorder.
 3. The method of claim 2, wherein themalignant B-cell lymphoproliferative disorder is Diffuse large B-celllymphoma, acute lymphocytic leukemia, lymphoid blastic phase ChronicMyeloid Leukemia, Chronic lymphocytic leukemia/Small lymphocyticlymphoma, Extranodal marginal zone B-cell lymphomas, Mucosa-associatedlymphoid tissue lymphomas, Follicular lymphoma, Mantle cell lymphoma,Nodal marginal zone B-cell lymphoma, Burkitt lymphoma, Hairy cellleukemia, Primary central nervous system lymphoma, Splenic marginal zoneB-cell lymphoma, Waldenstrom's macroglobulinemia/Lymphoplasmacyticlymphoma, Multiple myeloma, Plasma cells dyscrasias, Plasma cellneoplasms, Primary mediastinal B-cell lymphoma, Hodgkin Disease, orCastelman's Disease.
 4. The method of claim 3, wherein the malignantB-cell lymphoproliferative disorder is Diffuse large B-cell lymphoma. 5.The method of claim 4, wherein the Diffuse large B-cell lymphoma isDouble-Hit lymphoma.
 6. The method of claim 1, wherein the malignantlymphoproliferative disorder is a malignant T-cell lymphoproliferativedisorder.
 7. The method of claim 6, wherein the malignant T-celllymphoproliferative disorder is T-cell leukemia/lymphoma, Extranodalnatural killer/T-cell lymphoma, Cutaneous T-cell lymphoma,Enteropathy-type T-cell lymphoma, Angioimmunoblastic T-cell lymphoma,Anaplastic large T/null-cell lymphoma, Subcutaneous panniculitis-likeT-cell lymphoma, T-cell acute lymphocytic leukemia, T-cell largegranular lymphocyte leukemia, Lymphoid blastic phase Chronic MyeloidLeukemia, post-transplantation lymphoproliferative syndromes, humanT-cell leukemia virus type I-positive (HTLV-I+) adult T-cellleukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), orunspecified T-cell lymphoma.
 8. The method of claim 1, wherein themalignant lymphoproliferative disorder is chemotherapy-refractory. 9.The method of claim 1, wherein the 9-ING-41 is administered incombination with a second therapeutic agent.
 10. The method of claim 9,wherein the second therapeutic agent is administered in asub-therapeutic amount.
 11. The method of claim 9, wherein the secondtherapeutic agent is an anticancer agent.
 12. The method of claim 11,wherein the anticancer agent is an apoptosis modulator, a CDK modulator,or a modulator of the mTOR/AKT/PI3K pathway.
 13. The method of claim 12,wherein the anticancer agent is an apoptosis modulator.
 14. The methodof claim 13, wherein the apoptosis modulator is a Bcl-2 inhibitor. 15.The method of claim 14 wherein the Bcl-2 inhibitor is venetoclax,ABT-737, or navitoclax.
 16. The method of claim 15, wherein the Bcl-2inhibitor is venetoclax.
 17. The method of claim 12, wherein theanticancer agent is a CDK modulator.
 18. The method of claim 17, whereinthe CDK modulator is a CDK9 inhibitor.
 19. The method of claim 18,wherein the CDK9 inhibitor is BAY-1143572, LDC000067, Dinaciclib(SCH727965), SNS-032 (BMS-387032), AT7519, P276-00, AZD5438, PHA-767491,or PHA-793887.
 20. The method of claim 19, wherein the CDK9 inhibitor isBAY-1143572.
 21. The method of claim 12, wherein the anticancer agent isa modulator of the mTOR/AKT/PI3K pathway.
 22. The method of claim 21,wherein the modulator of the mTOR/AKT/PI3K pathway is a PI3K inhibitor.23. The method of claim 22, wherein the PI3K inhibitor is copanlisib oridelalisib.
 24. The method of claim 23, wherein the PI3K inhibitor isidelalisib.